1. Field of the Invention
With the advent of hybrid DNA technology and the explosion in the ability to isolate, purify, and assay a wide variety of natural products, both polypeptides and nucleic acids, there is an increasing need for rapid and efficient methods for preparing oligomers of amino acids and nucleic acids.
With nucleic acids, there is the need to synthesize sequences for use as linkers, adapters, synthetic genes and synthetic regulatory sequences, as well as probes, primers, and the like. While only small amounts of materials are required in the initial application, since these sequences can be cloned, it is very important that the sequences be substantially free of sequences which include errors, since such sequences could result in constructions which result in undesired products or results.
For the poly(amino acids) or polypeptides, there is substantial interest in being able to synthesize naturally occurring polypeptides for investigation of their physiological properties, for production of polypeptide fragments and natural products, where such fragments can be studied for their physiological properties, be used as haptens for the production of antibodies specific for a determinant site of interest, drug agonist or antagonist, or the like.
Many procedures have been developed for producing oligomers of nucleotides, amino acids or other naturally occurring monomers. These procedures for the most part rely on attaching the first monomer by a selectively cleavable linkage to a solid support. Each monomeric unit is then added sequentially, with each addition involving a number of chemical reactions.
At each stage during the synthesis of the oligomer, there is a small but finite probability that a number of chains will not be extended. Therefore, during the oligomerization, a large number of errors may be introduced, where sequences are produced having single or multiple omissions of monomers. At the completion of the sequence and separation from a support, the desired sequence will be contaminated with sequences closely approximating the desired sequence. These errors may then manifest themselves in a number of different ways, varying with whether a polynucleotide or polypeptide is being prepared. With polynucleotides, when the sequences are being cloned and used in various constructions, errors may have been introduced where the clone which is selected includes the erroneous sequence. Without prior oligomer purification during sequencing of the construct, the error may be retained leading to undesired products, suboptimum performance, or the like. With polypeptides, the erroneous sequence may lead to different physiological activity from the intended sequence, the formation of antibodies binding to sequences other than the sequence of interest and possibly providing for erroneous results, as a result of the varying binding response.
It has therefore become of increasing importance to be able to prepare sequences with an assurance that there is substantially no contamination of sequences approximating the desired sequence, which would lead to erroneous products or observation. By removing failure sequences during preparation, one may also avoid the need for subsequent purifications, such as electrophoresis, which can result in loss of material. Loss of material can be a serious problem when dealing with the very small amounts of materials synthesized in initial stages involving cloning or investigative polypeptides.
2. Description of the Prior Art
Matteucci and Caruthers, J. Am. Chem. Soc. (1981) 103:3185-3191, describe the use of phosphorchloridites in the preparation of oligonucleotides. Beaucage and Caruthers, Tetra. Lett. (1981) 22:1859-1862 and U.S. Pat. No. 4,415,732 describe the use of phosphoramidites in the preparation of oligonucleotides. Smith, ABL Dec. 1983) 15-24, describes automated solid phase oligodeoxyribonucleotide systhesis. See also the references cited therein. See also, Warner et al., DNA (1984) 3:401-411, whose disclosure is incorporated herein by reference.
Amidine protection of adenosine has been described by McBride and Caruthers, Tetra. Lett. (1983) 24:245 and Froehler and Matteucci, Nucl. Acids Res. (1983) 11:8031. Other blocking groups will be described in the description.